Diabetes mellitus (DM) is the sixth and stroke is the third leading cause of death in the United States. Although both experimental and clinical data have shown that diabetic hyperglycemia (HG) augments brain damage due to cerebral ischemia, the molecular mechanisms underlying this important, clinically relevant phenomenon are poorly defined. Preliminary studies suggest that HG enhances the superoxide production of mitochondrial origin and causes mitochondria! functional and morphological alterations in the early stage of reperfusion following a transient cerebral ischemia. HG-enhanced reactive oxygen species (ROS) formation may be caused by increased proton potential across the inner mitochondrial membrane due to excessive extrusion of protons donated from NADH and FADH2 through glucose metabolism. Mitochondrial uncoupling proteins (UCPs) dissipate the mitochondrial proton gradient by transporting H+ across the inner membrane, thereby stabilizing the mitochondrial membrane potential and reducing the formation of ROS. However, existing eveidence provids conflicting results to whether UCP2 is neuroprotective or neurodestructive. Our Central Hypothesis is that diabetic hyperglycemia enhances brain damage during stroke by overproducing ROS from the mitochondrial electron transport chain (ETC) due to increased extrusion of protons that perturbs AM^m. Accordingly, if the membrane potential is stabilized by upregulation of UCP2 the formation of ROS may be reduced and the diabetes-exacerbated stroke damage may be prevented. Using in vitro neuronal cultures that simulate in vivo diabetic and ischemic stroke conditions and an in vivo stroke model in Tg-UCP2, KO-UCP2 and genetic background matched C57BL/6J mice under both non- DM and DM conditions, the objectives of this study are to: 1) determine the pathways leading to ROS production after in vitro oxygen deprivation (OD) in cultured neurons mimicking in vivo diabetes and stroke conditions, 2) determine if deletion of the UCP2 gene augments and overexpression of UCP2 prevents HG- enhanced brain damage using both in vitro and in vivo ischemia models, and 3) determine the pathways by which UCP2 exerts its protective effects. Our successful achievement of this goal should significantly contribute to the current understanding of this clinical problem and may lead to the development of new therapeutic approaches, e.g. via manipulation of UCPs expression in the brain as a treatment for the disease